The present invention relates generally to enzyme preparations for therapeutic administration to the eyes of humans or other mammals, and more particularly to a method for utilizing one or more enzymes to treat disorders of the eye affecting the retina and/or the vitreous body of the mammalian eye.
In human beings, the anatomy of the eye includes a xe2x80x9cvitreous bodyxe2x80x9d which occupies approximately four fifths of the cavity of the eyeball, behind the lens. The vitreous body is formed of gelatinous material, known as the vitreous humor. Typically, the vitreous humor of a normal human eye contains approximately 99% water along with 1% macromolecules including: collagen, hyaluronic acid, soluble glycoproteins, sugars and other low molecular weight metabolites.
The retina is essentially a layer of nervous tissue formed on the inner posterior surface of the eyeball. The retina is surrounded by a layer of cells known as the choroid layer. The retina may be divided into an optic portion which participates in the visual mechanism, and a non-optic portion which does not participate in the visual mechanism. The optic portion of the retina contains the rods and cones, which are effectual organs of vision. A number of arteries and veins enter the retina at its center, and splay outwardly to provide blood circulation to the retina.
The posterior portion of the vitreous body is in direct contact with the retina. Networks of fibrillar strands extend from the retina and permeate or insert into the vitreous body so as to attach the vitreous body to the retina.
Diabetic retinopathy, trauma and other ophthalmological disorders sometimes result in rupture or leakage of retinal blood vessels with resultant bleeding into the vitreous humor of the eye (i.e., xe2x80x9cvitreous hemorrhagexe2x80x9d). Such vitreous hemorrhage typically manifests as clouding or opacification of the vitreous humor.
Vitreous hemorrhage is sometimes, but not always, accompanied by tearing or detachment of the retina. In cases where the vitreous hemorrhage is accompanied by a retinal tear or detachment, it is important that such retinal tear or detachment be promptly diagnosed and surgically repaired. Failure to promptly diagnose and repair the retinal tear or detachment may allow photoreceptor cells of the retina, in the region of the tear or detachment, to become necrotic. Such necrosis of the photoreceptor cells of the retina may result in loss of vision. Furthermore, allowing the retinal detachment to remain unrepaired for such extended period of time may result in further vitreous hemorrhage and/or the formation of fibrous tissue at the site of the hemorrhage. Such formation of fibrous tissue may result in the formation of an undesirable permanent fibrous attachment between the vitreous body and the retina.
The typical surgical procedure used for repair of retinal tears or detachment requires that the surgeon be able to look through the vitreous humor, to visualize the damaged region of the retina (i.e., xe2x80x9ctransvitreous viewing of the retinaxe2x80x9d). When vitreous hemorrhage has occurred, the presence of the hemorrhagic blood within the vitreous can cause the vitreous to become so cloudy that the surgeon is prevented from visualizing the retina through the vitreous. Such hemorrhagic clouding of the vitreous can take 6-12 months or longer to clear sufficiently to permit trans-vitreal viewing of the retina. However, in view of the potential complications which may result from delayed diagnosis or treatment of a retinal tear or detachment, it is generally not desirable to wait for such natural clearance of the hemorrhagic blood to occur.
Furthermore, even when the vitreous hemorrhage is not accompanied by retinal tear or detachment, it is often difficult to verify that retinal tear or detachment has not occurred, because the clouded vitreous prevents the physician from performing routine funduscopic examination of the retina. Moreover, the presence of hemorrhagic blood within the vitreous may significantly impair or completely obscure the patient""s vision through the affected eye, and will continue to do so until such time as the hemorrhagic blood has been substantially or fully cleared.
Thus, the presence of hemorrhagic blood within the vitreous body causes multiple clinical problems including a) inability to visually examine and diagnose the site and cause of the hemorrhage and/or any accompanying tear or detachment of the retina, b) full or partial impairment of vision in the affected eye, and c) impairment or prevention of the performance of trans-vitreal surgical procedures of the type of treatment typically utilized to repair the site of hemorrhage and/or to repair any accompanying retinal tear or detachment.
In cases where vitreous hemorrhage has resulted in substantial clouding or opacification of the vitreous, the treating physician may have the option to perform a procedure known as a vitrectomy, wherein all (or a portion of) the vitreous body is removed from the interior of the eye, and replaced with a clear liquid. The performance of this vitrectomy procedure is intended to allow the surgeon to perform the necessary retinal examination and/or surgical repair of the hemorrhage and any accompanying retinal tear or detachment. Such vitrectomy procedures are highly skill intensive, and are associated with several significant drawbacks, risks and complications. Among these drawbacks, risks and complications are the potential that the act of removing the vitreous will cause further detachment or tearing of the retina and/or that such removal of the vitreous will cause further hemorrhage from the already weakened retinal blood vessels.
In an effort to minimize the potential for causing further detachment or tearing of the retina during performance of the vitrectomy, it has previously been proposed in U.S. Pat. No. 5,292,509 (Hageman), to inject certain protease-free glycosaminoglycanase enzymes into the vitreous body, to cause the vitreous body to become uncoupled or xe2x80x9cdisinsertedxe2x80x9d from the retina, prior to removal of the vitreous body. Such disinsertion or uncoupling of the vitreous body is purported to minimize the likelihood that further tearing or detachment of the retina will occur as the vitreous body is removed. Examples of specific protease-free glycosaminoglycanase enzymes which may be used to bring about this vitreal disinsertion purportedly include: chondroitinase ABC, chondroitinase AC, chondroitinase B, chondroitin 4-sulfatase, chondroitin 6-sulfatase, hyaluronidase and B-glucuronidase.
Although hyaluronidase enzyme has been known to be usable for various ophthalmic applications, including the vitrectomy adjunct application described in U.S. Pat. No. 5,292,509 (Hageman), previously published studies have indicated that hyaluronidase is toxic to the retina and/or other anatomical structures of the eye when administered intravitreally at doses in excess of 1 IU, that is, at 15, 30, 50 and 150 IU of hyaluronidase. See, The Safety of Intravitreal Hyaluronidase; Gottlieb, J. L.; Antoszyk, A. N., Hatchell, D. L. and Soloupis, P., Invest Ophthalmol Vis Sci 31:11, 2345-52 (1990).
The ophthalmic toxicity of some hyaluronidase preparations has been confirmed by other investigators, who have proposed that such hyaluronidase preparations be used as a toxic irritant for causing experimentally-induced neovascularization of the eye, in animal toxicity models. See, An Experimental Model of Preretinal Neovascularization in the Rabbit; Antoszyk, A. N., Gottlieb, J. L., Casey, R. C., Hatchell, D. L. and Machemer, R., Invest Ophthalmol Vis Sci 32: 1, 46-51 (1991).
Unfortunately, it has not been previously known whether the reported therapeutic activities and toxicities of hyaluronidase are universally applicable to all hyaluronidase preparations, or whether such efficacies and/or toxicities are applicable only to hyaluronidase preparations containing certain excipient materials or to hyaluronidase enzymes derived from specific sources. This is an important consideration in view of the fact that the purity and characterization (e.g., molecular weight distribution) of the various hyaluronidase preparations used in the prior art may vary, depending on the source of the hyaluronidase and the solvents and/or other formulation components with which the hyaluronidase is combined.
The term xe2x80x9chyaluronidasexe2x80x9d is commonly used to describe a group of endo-B-glucuronidase enzymes which depolymerize certain mucopolysaccharides, such as hyaluronic acid. Myer, K. et al., The Enzymes; Vol. 4, 2d, Ed., pp 447, Academic Press, Inc., New York (1960).
Hyaluronidase causes hydrolysis of the endo-N-acetyl hexosaminic bonds of hyaluronic acid and of the chondroitin sulfate acids A and C, primarily to tetrasaccharide residues.
Significant evidence indicates that hyaluronidase enzymes derived from different sources differ in enzyme molecular weight distribution and in specific enzymatic activities. Such variability in molecular weight distribution and specific enzymatic activity are noteworthy considerations in view of the fact that hyaluronidase enzymes may be isolated from a variety of sources, including bovine testes, ovine testes, certain bacteria such as streptomyces and certain invertebrate animals such as leeches.
The Wydase(copyright) hyaluronidase preparation is reported to have been previously administered to the eyes of mammals for various clinical and experimental applications, including the treatment of glaucoma and the promotion of liquefaction of the vitreous body during vitrectomy procedures wherein the vitreous body is removed from the eye.
Although some hyaluronidase preparations have been reported to exhibit desirable therapeutic effects when injected into or administered topically to the eye, the potential toxicities of hyaluronidase and/or the thimerosal preservative are cause for concern regarding the safety of routine clinical administration of such preparations by intraocular injection.
Accordingly, there exists a need in the art for the formulation and development of a new hyaluronidase preparation which may be administered to the eye at dosage levels which are sufficient to bring about optimal therapeutic effects, but which do not cause ocular toxicity.
Additionally, in view of the above-discussed problems associated with the slowness of natural clearance of hemorrhagic blood from the vitreous body, there exists a need in the art for the elucidation and development of new methods and procedures for accelerating the clearance of hemorrhagic blood from the vitreous body of the eye so as to permit trans-vitreal viewing of the posterior aspect of the eye, including the retina, without the need for removal of the vitreous body (i.e., total or partial vitrectomy).
Additionally, there is a need for the prevention and treatment of various disorders of the mammalian eye which result from damage or pathology to the vascularization of the retina or which result in damage to the blood-retinal barrier.
An enzymatic method is provided for treating ophthalmic disorders of the mammalian eye. Prevention of neovascularization and the increased rate of clearance from the vitreous of materials toxic to retina is accomplished by administering an amount of hyaluronidase effective to liquefy the vitreous humor of the treated eye without causing toxic damage to the eye. Liquefaction of the vitreous humor increases the rate of liquid exchange from the vitreal chamber. This increase in exchange removes those materials and conditions whose presence causes ophthamological and retinal damage.
A method for inducing liquefaction of a vitreous humor to prevent a disorder of a mammalian eye, comprising the step of contacting with the vitreous humor of a mammalian eye an amount of hyaluronidase effective to liquefy the vitreous humor, whereby the disorder is prevented without causing toxic damage to the mammalian eye.
Preferably, the method is carried out for the purpose of treating proliferative diabetic retinopathy, age-related macular degeneration, amblyopia, retinitis pigmentosa, macular holes, or macular exudates, or cystoid macular edema. The hyaluronidase enzyme may be in a liquid solution, and the step of contacting of the enzyme with the vitreous humor comprises, injecting said liquid solution into the vitreous humor. The hyaluronidase is contacted with the vitreous in the absence of thimerosal.
In one embodiment, the hyaluronidase is contacted with the vitreous humor at a dose of 5-200 International Units. In another embodiment the hyaluronidase is contacted with the vitreous humor at a dose of 1 International Units. The hyaluronidase may be administered in multiple doses, and a single intravitreal injection may have an injectate volume of less than 100:1.
In another embodiment, the hyaluronidase is devoid of hyaluronic acid lysing activity having a molecular weight above approximately 100,000 when determined by 10% SDS PAGE electrophoresis. Also, the hyaluronidase is devoid of gelatinolytic activity having a molecular weight between approximately 60,000-100,000 when determined by 10% SDS PAGE electrophoresis. Further, the hyaluronidase enzyme is devoid of caseinolytic activity having a molecular weight above approximately 45,000 when determined by 10% SDS PAGE electrophoresis. Moreover, the hyaluronidase is devoid of hyaluronidase matter having a molecular weight above approximately 100,000 when determined by 4-20% SDS PAGE electrophoresis. Additionally, the hyaluronidase is devoid of hyaluronidase matter having a molecular weight between approximately 50,000-60,000 when determined by 4-20% SDS PAGE electrophoresis. Furthermore, the hyaluronidase is devoid of hyaluronidase matter having a molecular weight below approximately 20,000 when determined by 4-20% SDS PAGE electrophoresis.
In another embodiment, the hyaluronidase is prepared in a solution for injection which is free of thimerosal and which has a formulation comprising hyaluronidase up to 8000 IU, the lactose is at 5.0-130.0 mg, and the phosphate at 0.01-100.0 mmoles. In yet another embodiment, the hyaluronidase is prepared in a solution for injection which is free of thimerosal and which has a formulation comprising hyaluronidase at 6500 IU, lactose at 5.0 mg, and phosphate at 0.02 mmoles. In still another embodiment, the hyaluronidase is prepared in a solution for injection which is free of thimerosal and which has a formulation such that the hyaluronidase is at 500-1000 IU, the lactose is at 5.0-10.0 mg, and the phosphate is at 0.01-10.0 mmoles.
In another embodiment of the present invention, a method for treating a disorder of a mammalian eye comprises the step of contacting with a vitreous humor of said mammalian eye an amount of hyaluronidase effective to treat said disorder without causing toxic damage to said mammalian eye. Here, the vitreous humor is free of hemorrhagic blood that permits viewing of a retina of said mammalian eye. Moreover, the contacting step is practiced in the absence of vitrectomy.